Ellen S. Morgan

Heterogeneity of the angiogenic response induced in different normal adult tissues by vascular permeability factor/vascular endothelial growth factor.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is an angiogenic cytokine with potential for the treatment of tissue ischemia. To investigate the properties of the new blood vessels induced by VPF/VEGF, we injected an adenoviral vector engineered to express murine VPF/VEGF164 into several normal tissues of adult nude mice or rats. A dose-dependent angiogenic response was induced in all tissues studied but was more intense and persisted longer (months) in skin and fat than in heart or skeletal muscle (≤3 weeks). The initial response (within 18 hours) was identical in all tissues studied and was characterized by microvascular hyperpermeability, edema, deposition of an extravascular fibrin gel, and the formation of enlarged, thin-walled pericyte-poor vessels (“mother” vessels). Mother vessels developed from preexisting microvessels after pericyte detachment and basement membrane degradation. Mother vessels were transient structures that evolved variably in different tissues into smaller daughter vessels, disorganized vessel tangles (glomeruloid bodies), and medium-sized muscular arteries and veins. Vascular structures closely resembling mother vessels and each mother vessel derivative have been observed in benign and malignant tumors, in other examples of pathological and physiological angiogenesis, and in vascular malformations. Together these data suggest that VPF/VEGF has a role in the pathogenesis of these entities. They also indicate that the angiogenic response induced by VPF/VEGF is heterogeneous and tissue specific. Finally, the muscular vessels that developed from mother vessels in skin and perimuscle fat have the structure of collaterals and could be useful clinically in the relief of tissue ischemia.

Basophils Produce IL-4 and Accumulate in Tissues after Infection with a Th2-inducing Parasite

Using mice in which the eGfp gene replaced the first exon of the Il4 gene (G4 mice), we examined production of interleukin (IL)-4 during infection by the intestinal nematode Nippostrongylus brasiliensis (Nb). Nb infection induced green fluorescent protein (GFP)pos cells that were FcɛRIpos, CD49bbright, c-kitneg, and Gr1neg. These cells had lobulated nuclei and granules characteristic of basophils. They were found mainly in the liver and lung, to a lesser degree in the spleen, but not in the lymph nodes. Although some liver basophils from naive mice express GFP, Nb infection enhanced GFP expression and increased the number of tissue basophils. Similar basophil GFP expression was found in infected Stat6−/− mice. Basophils did not increase in number in infected Rag2−/− mice; Rag2−/− mice reconstituted with CD4 T cells allowed significant basophil accumulation, indicating that CD4 T cells can direct both tissue migration of basophils and enhanced IL-4 production. IL-4 production was immunoglobulin independent and only partially dependent on IL-3. Thus, infection with a parasite that induces a “Th2-type response” resulted in accumulation of tissue basophils, and these cells, stimulated by a non-FcR cross-linking mechanism, are a principal source of in vivo IL-4 production.

Ultrastructural Evidence for Piecemeal and Anaphylactic Degranulation of Human Gut Mucosal Mast Cells in vivo

One hundred and seventeen coded intestinal biopsies were examined by electron microscopy and evaluated for morphological evidence of mast cell and basophil secretion in situ. Sixty percent of the biopsies had evidence of secretion. Mast cell secretion was evident in control biopsies, many of which were obtained from uninvolved tissues of patients with inflammatory bowel disease. Biopsies of inflamed continent pouches from ulcerative colitis (UC) patients showed more mast cell secretion than noninflamed UC pouch biopsies. This evidence of mast cell secretion supports recent work that documents high constitutive levels of histamine in jejunal fluids of Crohns disease patients and suggests a proinflammatory role for mast cells in inflammation associated with pouchitis.

Ultrastructural immunogold localization of prostaglandin endoperoxide synthase (cyclooxygenase) to non-membrane-bound cytoplasmic lipid bodies in human lung mast cells, alveolar macrophages, type II pneumocytes, and neutrophils.

Lipid bodies are non-membrane-bound, lipid-rich cytoplasmic inclusions that occur in many mammalian cell types. Because lipid bodies are more prominent in cells associated with inflammation and are repositories of arachidonyl-phospholipids, a role for lipid bodies in the oxidative metabolism of arachidonic acid to form eicosanoids has been suggested. To evaluate further whether lipid bodies, in addition to serving as non-membranous sources of substrate arachidonate, are involved in eicosanoid formation, we used cells isolated from human lung to investigate the intracellular localization of prostaglandin endoperoxide (PGH) synthase (cyclooxygenase), the key initial, rate-limiting enzyme in the formation of prostaglandins and thromboxanes. Isolated lung cells containing a mixture of mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils from short-term cultures were fixed in suspension in a dilute aldehyde mixture, post-fixed in osmium tetroxide, stained en bloc with uranyl acetate, dehydrated in a graded series of alcohols, and embedded in Epon. A post-embedding immunogold procedure was used with a primary PGH synthase monoclonal antibody and 20-nm gold-conjugated secondary antibody to demonstrate enzyme locations. Specificity controls were also done. We found PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils. Specific secretory and lysosomal granules and plasma membranes did not express PGH synthase. Specificity controls, including omission of the primary antibody or substitution with an irrelevant antibody, were negative. Absorption of the specific PGH synthase antibody with purified solid-phase PGH synthase resulted in a marked reduction of label in lipid bodies of all four cell types. These findings establish the presence of PGH synthase in lipid bodies of human lung mast cells, alveolar macrophages, Type II alveolar pneumocytes, and neutrophils and, in concert with previous studies, suggest that these cytoplasmic lipid-rich organelles may be non-membrane sites of eicosanoid formation.

Loss of histochemical identity in mast cells lacking carboxypeptidase A

ABSTRACT Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa− / − mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa − / − peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa − / − peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa − / − mice. The Mc-cpa − / − mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells.

Ultrastructural identification of exocytosis of granules from human gut eosinophils in vivo.

Twenty-two percent of 117 biopsies of human intestinal tissues had ultrastructural images of classical regulated secretion from eosinophils in vivo i.e. eosinophil granule extrusion (EGE). Replicate intestinal biopsies that were positive for bacteria had EGE more often than not (p < 0.05); 77% of the isolates were Staphylococci. Some of the intestinal biopsies also had damaged nerves; all that had EGE and damaged enteric nerves also had positive bacterial cultures. The EGE that we observed could not account for all enteric nerve damage, suggesting multifactorial mechanisms for nerve damage in gut tissues. Among the possibilities are release of neurotoxic eosinophil granule proteins by an alternate secretory route, i.e., piecemeal degranulation, direct toxicity of tissue invasive bacteria and/or damaged nerves of unknown etiology such as those that are regularly present in uninvolved tissues of patients with Crohns disease.

Human Gut Mucosal Mast Cells: Ultrastructural Observations and Anatomic Variation in Mast Cell-Nerve Associations in vivo

One hundred and seventeen coded intestinal biopsy specimens were examined by electron microscopy. All surgical biopsies were obtained from uninvolved sites of patients with two inflammatory bowel diseases (ulcerative colitis or Crohns disease) and from patients with preneoplastic and neoplastic diseases (adenocarcinoma, rectal polyp, familial polyposis). Biopsy sites included normal ileum, colon, and rectum as well as conventional ileostomies and continent pouches constructed from the ileum. The data reported here describe the ultrastructural anatomy of human gastrointestinal tract mucosal mast cells in vivo and their anatomic associations with enteric nerves.

Ultrastructural Localization of Prostaglandin Endoperoxide Synthase (Cyclooxygenase) to Isolated, Purified Fractions of Guinea Pig Peritoneal Macrophage and Line 10 Hepatocarcinoma Cell Lipid Bodies

Subcellular fractions of purified cytoplasmic, nonmembrane-bound lipid bodies were prepared from [3H]-arachidonic acid-labeled guinea pig peritoneal macrophages and line 10 hepatocarcinoma cells. These fractions, which contained [3H]-arachidonyl lipids, were shown to be devoid of contaminating cellular membranes by electron microscopy, and to contain prostaglandin endoperoxide (PGH) synthase by postembedding immunogold electron microscopy. These findings support a proposed role for these lipid-rich organelles in the generation of eicosanoids by oxidative metabolism of arachidonate in the cyclooxygenase pathway of inflammatory and neoplastic cells.

Vesicle-mediated secretion of human eosinophil granule-derived major basic protein

Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.

Axonal necrosis of enteric autonomic nerves in continent ileal pouches. Possible implications for pathogenesis of Crohn's disease.

OBJECTIVE Axonal necrosis was first described in samples of small intestine from patients with Crohns disease (A.M. Dvorak et al. Hum Pathol 1980; 11:620-634). Clinically evident inflammation of continent ileal reservoirs (pouches) has clinical features that resemble Crohns disease. Possible similarities in the pathogenesis of Crohns disease and pouchitis were sought using ultrastructural and microbiologic tools to identify damaged enteric nerves and tissue bacteria. METHODS An encoded ultrastructural and microbiologic study of replicate biopsies from 114 samples of human intestine was done. Biopsies from ileum, colon, conventional ileostomy or continent pouch were obtained from patients with ulcerative colitis, Crohns disease, or familial polyposis and grouped into three clinical study groups (control, normal pouch, pouchitis), based on clinical and endoscopic criteria. Biopsies were prepared for electron microscopy with standard methods; replicate biopsy samples were washed extensively before preparing cultures designed to identify aerobic as well as facultative and obligate anaerobic bacteria (Onderdonk et al. J Clin Microbiol 1992; 30:312-317). The ultrastructural diagnosis of damaged enteric nerves was based on previously published criteria for axonal necrosis (A.M. Dvorak and W. Silen. Ann Surg 1985; 201:53-63). Intergroup comparisons were tested for significance using Chi-square analysis. RESULTS The highest incidence of axonal necrosis was present in Crohns disease control biopsies (53%), regardless of whether bacteria were present (or not) in cultures of replicate biopsies. Axonal necrosis also occurred in more ulcerative colitis and familial polyposis biopsies (regardless of biopsy site) that had positive bacterial cultures than in those that did not (p < 0.001). In addition, axonal necrosis was documented in 42% of the pouch biopsies from ulcerative colitis and familial polyposis patients, particularly in those pouches that were found to be inflamed by clinical criteria and that also had positive bacterial cultures of the biopsied tissues. Control biopsies from patients with ulcerative colitis and familial polyposis had significantly less nerve damage than pouch biopsies in the presence of positive cultures (p < 0.01). Among the clinically inflamed pouches biopsied in ulcerative colitis or familial polyposis patients, we found that none had damaged enteric nerves when bacterial cultures were negative (p < 0.005). If the presence of axonal necrosis alone was compared with the presence of undamaged enteric nerves in all biopsies from patients with ulcerative colitis, a highly significant number of ulcerative colitis biopsies with axonal necrosis occurred in pouches (72%) compared with controls (p < 0.001). CONCLUSIONS The ultrastructural finding of axonal necrosis in Crohns disease confirms previous studies. The presence of damaged enteric nerves in patients with pouchitis provides ultrastructural support to the clinical impression of similarities between pouchitis and Crohns disease. The association of damaged nerves and invasive bacteria in pouchitis suggests mechanistic similarities for the pathogenesis of Crohns disease that requires further investigation.